HAEMATOLOGICAL RESPONSE OF NILE TILAPIA (Oreochromis niloticus)
JUVENILES EXPOSED TO TOBACCO (Nicotiana tobaccum) LEAF DUST
M.O. OLUFAYO AND I.A. JATTO
Department Of Fisheries and Aquaculture Technology, Federal University of Technology, Akure
ABSTRACT
Tobacco (Nicotiana tobaccum) leaf dust has piscicidal properties thus there is a need to study its
haematological effects on Nile tilapia juveniles. A 96hour bioassay was conducted on Oreochromis
niloticus juveniles (mean wt., 30g) to determine the median lethal concentrations (LC50) .The fish
were exposed to various concentrations of tobacco leaf dust (0.5g-2.5g/l).Water quality parameters
and physiological parameters were monitored/determined according to standard procedures. Water
quality parameters were monitored after 96hours. The LC50 at the end of 96hours was 1.35g/l. The
monitored water quality parameters such as temperature, pH and dissolved oxygen were significantly
decreased while total alkalinity and conductivity increased significantly in the exposed media,
compared to the control test. The fish showed hypeventilation, erratic swimming, loss of reflex during
the period of exposure and this increased with increase concentrations of tobacco leaf dust.
Haematological analysis of the blood revealed significant haematological changes, the intensity of
haematology damages increased with increasing concentrations and exposure to tobacco leaf dust.
The reduction in blood parameters could be as a result of destruction of erythrocyte or haemodilution.
However, the monitored water quality parameters revealed that the plant dust has effects on the
blood parameters of the test fish and consequently the biodiversity of the organisms. The result
provided baseline information and established safe limits of using tobacco leaf dust in fish ponds,
hence 1.0g/l concentration of tobacco leaf dust is recommended for the use on O. niloticus juveniles
INTRODUCTION
Aquaculture is increasingly becoming one of the fastest growing aspect of the agricultural industry
worldwide (FAO, 2004). Fish farmers often use tobacco leaf in controlling these unwanted
organisms/pests (Konar, 1970; Tobor, 1990). According to Aleem (1987), the use of tobacco leaf dust
is due to its inexpensiveness, local availability and easier degradability. Despite, the effective use of
this plant material, eco-toxicologists are interested in the ecotoxic properties of plant origin
pesticides/piscicides, such that plant origin pesticides/piscicides cannot be used directly in freshwater
bodies unless their toxicity and sub-lethal long term effect have been studied on non-target animals,
sharing the habitat with the target animals.
The active ingredient of the plant used, is the nicotine (Hassal, 1982), It is soluble in water,
alcohol, chloroform, ether, kerosene and some fixed oils (Vogue, 1984). Tobacco leaf dust has been
used in Nigeria as an effective insecticides and treatment of predators/pest in water (pond) since it is
completely biodegradable (Aleem, 1987; Nile tilapia is of the commercially important species of fish for
rapid aquaculture expansion in Nigeria. The choice of the test fish is attributed to the report of Rand
et al., (1995) that in order to extrapolate meaningful, relevant and ecological significant results from
aquatic toxicity tests, not only appropriate test but also appropriate organism should be used,
whenever possible, species should be studied or representative of the ecosystem that may be
impacted; thus the choice of the Oreochromis niloticus which is of economic importance in Nigeria as
an abundant cultural fish species in Nigeria and is very popular with fish farmers and consumers. The
knowledge of sub-lethal effects of tobacco is very important to delineate the health of fish status and
to provide a future understanding of ecological impacts (Radhaiah et al., 1987). The aim of this
research is to ascertain the assumption whether tobacco leaf dust ( Nicotina tobaccum) in a sub-lethal
concentration and in a medium exposure time can influence changes in the blood of O. niloticus after
the 96 hours exposure period.
MATERIALS AND METHODS
Juvenile O. niloticus of the same brood stock (30.01±0.34g) were obtained from the Federal
University of Technology, Akure fish farm. They were acclimatized in a glass tank for 24 hours. The
mortality and later transferred to the experimental plastic aquaria 10 fish/48L aquaria). The leaves of
tobacco were sun-dried for 10 days and milled into powder, sieved and stored in a sealed plastic
container until required. The concentrations of tobacco used were calculated as 50% 96h LC50 (96h
LC50 of tobacco leaf dust on O. niloticus obtained from preliminary investigation). Thus 100 mg of
tobacco leaf dust were measured and mixed in 1 litre of water to give 100 mg/L concentration of the
tobacco leaf dust. These concentrations were introduced into 12 sets of aquaria with one replication.
Forty (48) liters capacity aquaria were maintained throughout the exposure period. Ten (10)
juveniles each were placed in the 48L plastic aquarium. Bore-hole water was used during the
acclimatization and exposure period. In order to monitor the toxicant strength, level of dissolved
oxygen, the effects of evaporation; ammonia concentration and reduce stress during experimentation,
the test media were replaced by 50% prepared – concentrations of the same quantity after removing
its equivalent along with defaecation every 6 hours to maintain the requisite level and potency of the
concentration. The exposure period lasted for 96hours during which some water quality parameters
were monitored daily using APHA (1998) methods. After 96 hours, 60 fishes were sacrificed and
analyzed for the haematological examination. Blood was obtained from randomly selected fish from
the control and the exposed test after the 96hours, using 2.0ml plastic syringe, as described by KoriSiakpere (1998). The blood was transferred into a lithium heparin anticoagulant tube at room
temperature for 30-40 minutes (Mahoba, 1987) and stored at refrigerator until analyses.
Fish mortality data were analyzed using complete randomized design with equal replication
(one-way ANOVA test) at 5% level probability. All data were presented as means ± standard error,
the data from the 96hours tobacco leaf dust exposure was first analyzed using a one-way ANOVA test,
after which individual means were compared, using Bonferoni multi-sample correction/test.
RESULTS AND DISCUSSION
Mean values of water quality parameters of the different sub-lethal concentrations of tobacco leaf dust
and control media to which the test fish O. niloticus were exposed over the 96hours exposure period
is as presented in Table1. The value of temperature, pH and dissolved oxygen were found to
significantly (p<0.05) and (p<0.01) decreased as the concentrations of tobacco leaf dust increased.
However, the values of total alkalinity and conductivity in the exposed media were significantly
(p<0.01) increased as the concentrations of tobacco leaf dust increased, compared to the control test.
Exposure of O. niloticus juveniles to tobacco leaf dust solution clearly disrupted haematological
parameters. Haematocrit, haemoglobin values, erythrocyte and leucocyte counts, total protein and
albumin of the fish exposed to different concentrations of tobacco leaf dust revealed significant
haematological alteration and changes (Table 2). Erythrocyte reduces from mean value of 1.67 –
1.0mm3 with increase in concentration of tobacco leaf dust, the decrease in these values were also
observed to be both a factor of time and concentration of tobacco.
Table 1. Water quality parameters of the sub-lethal concentrations of tobacco leaf dust after 96 hours
Concentration
(g/l)
Temperature
(0C)
0.0
24.20 ±0.00
0.5
24.30 ±0.00
1.0
24.30 ±0.00
1.5
24.30 ±0.00
2.0
24.30 ±0.00
2.5
24.30 ±0.00
*mean ± Standard Error (SE).
Dissolved
oxygen
(mg/l)
6.20±0.10
5.90±0.20
4.10±0.10
3.80±0.10
3.30±0.00
3.10±0.10
pH
Conductivity
6.80±0.05
6.50±0.00
6.40±0.00
6.40±0.05
6.30±0.05
6.20±0.00
117.9±0.40
134.3±1.10
141.0±1.20
145.8±2.05
150.1±1.05
Table 2: Blood parameters of O. niloticus exposed to tobacco leaf dust concentrations for 96 hours.
Concentrations
(mg/l)
0.0
PCV
(%)
15.00
(1.00)
0.5
14.00
(1.00)
1.0
11.7
(0.58)
1.5
10.7
(0.58)
2.0
9.33
(1.16)
2.5
9.00
(1.00)
*Mean ± Standard Error
Haematological Parameters
RBC
WBC
HB
Total protein
(mm3) (mm3) (g/dl) (mg/dl)
1.67
(0.27)
1.73
(0.06)
1.21
(0.04)
1.21
(0.01)
1.10
(0.03)
1.00
(0.03)
(SE).
3.88
(0.10)
3.82
(0.10)
5.12
(0.10)
5.00
(0.10)
5.12
(0.08)
5.27
(0.76)
6.93
(2.57)
4.67
(0.31)
4.00
(0.00)
2.20
(0.35)
1.33
(0.35)
0.70
(0.10)
5.13
(0.12)
4.80
(0.20)
3.43
(0.21)
2.80
(0.20)
2.20
(0.20)
1.97
(0.15)
Albumin
(mg/dl)
3.43
(0.21)
2.93
(0.12)
2.73
(0.12)
2.37
(0.32)
2.00
(0.20)
1.87
(0.23)
Haemoglobin values reduced from 6.93-0.70g/dl with increase in concentration from 0.0g /l
control - 2.5g/ l (Table2). The erythrocyte and leucocyte counts showed intra- concentration
variations, the number of the leucocytes increased as the concentration of the toxicants increases
while erythrocytes deceased with increasing concentrations of tobacco leaf dust , In different
concentrations of tobacco leaf dust (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5g/l), PCV values varied from 15.0
,14.00, 11.67 ,10.67, 9.33 and 9.0%, respectively, at concentrations 0.5 and 2.5g/l, and the PCV
values reduced with increase in concentration of test dye and this is traceable to different fishes
having different blood parameters unlike human blood that is constant (Baker and Silverton, 1982).
Total protein (g/L) varied with different concentrations of tobacco leaf dust, the mean value for total
protein varies from 1.97-4.80mg/dl and 1.87-3.43mg/dl in albumin, this shows that total protein
decreases with increase in concentration of tobacco leaf dust when compared with the control (0.0)
with mean value of 5.13mg/dl (Table2). The 96-h LC50 was 1.35 g/L tobacco leaf extract, compared to
other synthetic pesticides used in fish farming, such as carbamates and organophosphates, tobacco
based products are certainly less toxic to fish (Wan et al., 1996). Results indicated that tilapia is more
sensitive to tobacco leaf water extract than other fishes (Mamdouh et al., 2008). Changes in the water
quality parameters showed that the concentrations affected the water quality, but the values were
within tolerance range (Table 1).
Houston et al. (1971) reported changes in the blood parameters of fresh water fish exposed
to various handling procedure before experiment and the effect of stress on the fish. The disrupted
haematological parameters observed in this experiment also agreed with Akinbulumo (2005) who
reported that fish showed toxic reaction to Derris elliptica root powder by surfacing jaws and
becoming stupefied. Reduction in oxygen level in this study is in line with Lloyd (1961) who reported
that toxicity of several poison on rainbow trout increased inversely to the oxygen concentrations of
water .A number of poisons become more toxic at low oxygen concentrations because of an
increasing respiratory rate, increasing the amount of poison to which the animal is exposed.
Haematological examination revealed adverse effect of tobacco leaf dust on the blood of O.
niloticus, and this is similar to Mason et al. (1994) who had earlier reported similar observations when
subjected O. niloticus to sub-lethal concentration of formalin. The result from statistical analysis shows
that there is reduction in some of the blood parameters, this is an indication of anemia which is a
condition characterized by a deficiency of haemoglobin, packed cell volume and erythrocytes.
REFERENCES
Akinbulumo, M .O (2005) : Derris elliptica as anaesthetic agent on Nile Tilapia,Oreochromis niloticus (
Linne, 1758), Applied Tropical Agriculture, 10, special issue: 24 -27.
Aleem, SO. 1987. An assessment of tobacco waste for control of the gastropod Tympandomus fuscavis
(Linnaeus) in brackish water fish pond. Aquaculture 73: 19-25.
Baker, F.J. and Silverton R.E.(1982): Introduction to Medical Lab Tech., 5 th Ed. Butterworth Scientific
London.
FAO. 2004. FAO Fisheries circular 886. Rev.25-29 June Gill R Foster A and Woodruff G. 1988. MK – 801
is neuroprotective in gerbils when administered during the post-ischemic period. Neuroscience
25: 847 – 855.
Hassal KA. 1982. The chemistry of pesticides; Macmillan press, London, 372p. Hill AB. 1995. The
environment and disease, Association or Causation? Proceedings of the Royal Society of
Medicine pp.295-300
Houston, A.M., Madden, J.A. Wood, R.J. and Miles, H.M.(1971) Variation in the blood and tissue
chemistry of Brook trout, Salvelinus fontinalis subsequent. Journal of the Fisheries Research
Board of Canada, 28 (5): 635-642:
Konar SK. 1970. Nicotine as a fish poison. Te Progressive Fish Culturist, 32: 103-104
Kori-Siakpere O. 1998. Petroleum induced alterations in the haematological parameters of Clarias
gariepinus. Nigerian Journal of Science and Environmental, 1:87-92
Mahoba GP. 1987. Studies on Indian Cichlids Ph.D thesis, University of Science and Technology,
Cochin, India
Lloyd, R. (1961): Effect of dissolved oxygen concentration on the toxicity of several poison of Exp. Bio.
38: 447-455
Mason, C.F. (1998): Biology of Freshwater pollution,Third edition, Longman, England. 356pp
Radhaiah V Girija M and Rao KJ. 1987. Changes in selected biochemical parameters In the kidney and
blood of the fish, Tilapia mossambicus (Peters), exposed to heptachlor. Bulletin of Environmental
Contamination and Toxicology, 39: 1006-1011.
Rand GM Wells PG. and McCarthy LS. 1995. Introduction to aquatic toxicology. In: G.M. Rand (ed),
Fundamental of aquatic Toxicology: effects environmental fate and risk assessment. 2nd ed.
Taylor and Francis, Washington, D.C
Tobor JG. 1990. The Fishing Industry in Nigeria: Status and potential for self sufficiency in fishing
production, NIOMR, Technical Paper, No.54 Nigeria Institute for Oceanography Research, Lagos,
Nigeria, 26pp.
Vogue E. 1984. Tobacco Encylopaedia, Tobacco Journal International Federal Republic of Germany.
Wan, M.T., R.G. Watts, M.B. Isman and R. Strub. 1996. Evaluation of the acute toxicity to juvenile
pacific northwest salmon of azadirachtin, neem extract, and neem based products. Bull. Environ.
Contam. Toxicol., 56: 432–439.
Mamdouh, A. A. Mousa, Ahmed, M. M. EL-Asham, and Mona Hamed. 2008. Effect of Neem leaf extact
on freshwater fishes and zooplankton community.
Aquaculture. 2008, Pp11.
8th International symposium on Tilapia